Isolation of Cellulolytic Anaerobic Extreme Thermophiles from New Zealand Thermal Sites

Avicel enrichment cultures from 47 thermal-pool sites in the New Zealand Rotorua-Taupo region were screened for growth and carboxymethyl cellulase activity at 75°C. Eight anaerobic cellulolytic cultures were obtained. The effect of temperature on carboxymethyl cellulase activity was measured, and bacteria were isolated from the five best cultures. Bacteria from two sources designated TP8 and TP10 grew at 75°C, accumulated reducing sugar in the growth medium and gave free cellulases with avicelase activity. Bacteria from sources designated Tok4, Tok8, and Wai21 grew at 75°C, accumulated no free sugars in the medium, and gave free carboxymethyl cellulases with virtually no avicelase activity. All were obligate anaerobic nonsporeforming rods which stained gram negative, grew on pentoses as well as hexoses, and gave ethanol and acetate as major fermentation end products. The isolated strain which produced the most active and stable cellulases (trivially designated TP8.T) had lower rates of free endocellulase accumulation at 75°C than did Clostridium thermocellum at 60°C, but its cellulase activity against avicel and filter paper in culture supernatants was comparable. Tested at 85°C, TP8.T carboxymethyl cellulases included components which were very stable, whereas C. thermocellum carboxymethyl cellulases were all rapidly inactivated. The TP8.T avicelase activity was relatively unaffected by Triton X-100, EDTA, and dithiothreitol. Evidence was obtained for the existence of unisolated, cellulolytic extreme thermophiles producing cellulases which were more stable and active than those from TP8.T.     

Anaerobic Growth,a Property Horizontally Transferred by an Hfr-Like Mechanism among Extreme Thermophiles

Despite the fact that the extreme thermophilic bacteria belonging to the genus Thermus are classified as strict aerobes, we have shown that Thermus thermophilus HB8 (ATCC 27634) can grow anaerobically when nitrate is present in the growth medium. This strain-specific property is encoded by a respiratory nitrate reductase gene cluster (nar) whose expression is induced by anoxia and nitrate (S. Ramírez-Arcos, L. A. Fernández-Herrero, and J. Berenguer, Biochim. Biophys. Acta, 1396:215–1997). We show here that this nar operon can be transferred by conjugation to an aerobic Thermus strain, enabling it to grow under anaerobic conditions. We show that this transfer takes place through a DNase-insensitive mechanism which, as for the Hfr (high frequency of recombination) derivatives of Escherichia coli, can also mobilize other chromosomal markers in a time-dependent way. Three lines of evidence are presented to support a genetic linkage between nar and a conjugative plasmid integrated into the chromosome. First, the nar operon is absent from a plasmid-free derivative and from a closely related strain. Second, we have identified an origin for autonomous replication (oriV) overlapping the last gene of the nar cluster. Finally, the mating time required for the transfer of the nar operon is in good agreement with the time expected if the transfer origin (oriT) were located nearby and downstream of nar.

Most extreme thermophiles that live in geothermal environments are strict anaerobes (3, 11) as a consequence of the adaptation to the low solubility of oxygen at these temperatures. However, members of the genus Thermus constitute an exception to this general rule, being described taxonomically as strictly aerobic chemorganotrophs (2).However, we recently showed that one of the most thermophilic isolates of this genus, Thermus thermophilus HB8, was able to grow anaerobically when nitrate was present in the medium. Biochemical and genetic evidence demonstrated that this ability was related to the synthesis of a membrane-bound respiratory nitrate reductase complex whose protein components, the α (NarG; 136 kDa), β (NarH; 57 kDa), and γ (NarI; 28 kDa) subunits, were homologous (about 48 to 50% sequence identity) to those from mesophilic facultative anaerobes (e.g., Escherichia coli). The genes encoding these subunits were located within a single operon (nar) that was induced under low oxygen concentrations when nitrate was present (21). In contrast to those described for most nitrate reducers, the product of nitrate respiration was secreted to the growth medium through an unknown transporter.We also observed that even a closely related strain, such as T. thermophilus HB27, was unable to grow under such anaerobic conditions (21). Since the main difference between strains HB8 and HB27 of T. thermophilus is the absence of plasmids from the latter, the possibility that the nar operon could be encoded by a transferable genetic element, such as a plasmid, was considered.In this article, we analyze this possibility and demonstrate that the ability to grow by nitrate respiration can be transferred to the aerobic strain T. thermophilus HB27 by conjugation. We also relate this ability to the integration of a nar-carrying conjugative plasmid into the chromosome of T. thermophilus HB8. Moreover, we show that, as for the Hfr strains of E. coli, this integrated plasmid can also mobilize other chromosomal genes in a time-dependent way.     

Effect of Temperature on the Fatty Acid Composition of the Extreme Thermophiles, Bacillus caldolyticus and Bacillus caldotenax

Gas chromatographic analysis of extracts from Bacillus caldolyticus and Bacillus caldotenax grown at 60, 70, or 80 C showed that both contain branched-chain fatty acids as major constituents at all temperatures tested. With increasing temperature, a decrease of i-C15 and an increase of i-C17 fatty acids were observed in both strains, as well as a decrease of i-C16 fatty acids corresponding to an increase of n-C16 fatty acids. The most obvious difference was that the shifts observed with B. caldolyticus occurred mainly upon raising the temperature to 10 C above, and in B. caldotenax upon lowering the temperature to 20 C below, the optimal growth temperature.     

Microtubule Stabilization in Pressure Overload Cardiac Hypertrophy

Increased microtubule density, for which microtubule stabilization is one potential mechanism, causes contractile dysfunction in cardiac hypertrophy. After microtubule assembly, α-tubulin undergoes two, likely sequential, time-dependent posttranslational changes: reversible carboxy-terminal detyrosination (Tyr-tubulin ↔ Glu-tubulin) and then irreversible deglutamination (Glu-tubulin → Δ2-tubulin), such that Glu- and Δ2-tubulin are markers for long-lived, stable microtubules. Therefore, we generated antibodies for Tyr-, Glu-, and Δ2-tubulin and used them for staining of right and left ventricular cardiocytes from control cats and cats with right ventricular hypertrophy. Tyr- tubulin microtubule staining was equal in right and left ventricular cardiocytes of control cats, but Glu-tubulin and Δ2-tubulin staining were insignificant, i.e., the microtubules were labile. However, Glu- and Δ2-tubulin were conspicuous in microtubules of right ventricular cardiocytes from pressure overloaded cats, i.e., the microtubules were stable. This finding was confirmed in terms of increased microtubule drug and cold stability in the hypertrophied cells. In further studies, we found an increase in a microtubule binding protein, microtubule-associated protein 4, on both mRNA and protein levels in pressure-hypertrophied myocardium. Thus, microtubule stabilization, likely facilitated by binding of a microtubule-associated protein, may be a mechanism for the increased microtubule density characteristic of pressure overload cardiac hypertrophy.     

Artificial Environments for the Co-Translational Stabilization of Cell-Free Expressed Proteins

An approach for designing individual expression environments that reduce or prevent protein aggregation and precipitation is described. Inefficient folding of difficult proteins in unfavorable translation environments can cause significant losses of overexpressed proteins as precipitates or inclusion bodies. A number of chemical chaperones including alcohols, polyols, polyions or polymers are known to have positive effects on protein stability. However, conventional expression approaches can use such stabilizing agents only post-translationally during protein extraction and purification. Proteins that already precipitate inside of the producer cells cannot be addressed. The open nature of cell-free protein expression systems offers the option to include single chemicals or cocktails of stabilizing compounds already into the expression environment. We report an approach for systematic screening of stabilizers in order to improve the solubility and quality of overexpressed proteins co-translationally. A comprehensive list of representative protein stabilizers from the major groups of naturally occurring chemical chaperones has been analyzed and their concentration ranges tolerated by cell-free expression systems have been determined. As a proof of concept, we have applied the method to improve the yield of proteins showing instability and partial precipitation during cell-free synthesis. Stabilizers that co-translationally improve the solubility and functional folding of human glucosamine 6-phosphate N-acetyltransferase have been identified and cumulative effects of stabilizers have been studied.     

Stabilization of Soluble Proteins in Vitro by Heat Shock Proteins-Enriched Ammonium Sulfate Fraction from Soybean Seedlings

The 70-100% ammonium sulfate fraction of postribosomal supernatantof heat shocked soybean seedlings contained a high percentageof all of the heat shock proteins. The proteins in this fractionwere resistant to heat denaturation, as judged by their unpelletabilityafter heat treatment. Moreover, this fraction, when added tothe postribosomal supernatant from control (non-heat shocked)seedlings, showed a significant ability to protect the controlproteins from heat denaturation. Heated at 55°C, some 50%of the control proteins, which were normally denatured afterheat treatment, were protected for at least 1 h when heat shockproteins-enriched fraction was added. The degree of protectionwas proportional to the amount of heat shock proteins-enrichedfraction added. However, when the ammonium sulfate fractionprepared from the seedlings with a heat treatment at 40°Cfor 3 h followed with a brief heat shock at 45°C which depletedmost of the 15–18 kDa and partial 68–70 kDa, 24kDa and 22 kDa heat shock proteins was added the effectivenessin preventing heat denaturation was lost. This suggests thatthe heat shock proteins of 15–18 kDa with those of 68–70kDa and perhaps 24 kDa and 22 kDa are important for providingthe protection from heat denaturation. (Received July 28, 1988; Accepted March 2, 1989)     

An Unusual Hydrophobic Core Confers Extreme Flexibility to HEAT Repeat Proteins

Alpha-solenoid proteins are suggested to constitute highly flexible macromolecules, whose structural variability and large surface area is instrumental in many important protein-protein binding processes. By equilibrium and nonequilibrium molecular dynamics simulations, we show that importin-β, an archetypical α-solenoid, displays unprecedentedly large and fully reversible elasticity. Our stretching molecular dynamics simulations reveal full elasticity over up to twofold end-to-end extensions compared to its bound state. Despite the absence of any long-range intramolecular contacts, the protein can return to its equilibrium structure to within 3 Å backbone RMSD after the release of mechanical stress. We find that this extreme degree of flexibility is based on an unusually flexible hydrophobic core that differs substantially from that of structurally similar but more rigid globular proteins. In that respect, the core of importin-β resembles molten globules. The elastic behavior is dominated by nonpolar interactions between HEAT repeats, combined with conformational entropic effects. Our results suggest that α-solenoid structures such as importin-β may bridge the molecular gap between completely structured and intrinsically disordered proteins.     

Lysozyme Stabilization under High Pressure: Differential Scanning Microcalorimetry

The heat denaturation of lysozyme has been studied by high-pressure differential scanning microcalorimetry. It has been demonstrated that an increase in pressure has different influence on denaturation temperature and enthalpy at different pH values. It has been established that the pressure increase has no appreciable effect on the transition cooperativity. The experimental data have been analyzed using an equilibrium model of transition between two states. Partial molar volume changes accompanying the denaturation as well as isothermal compressibility and thermal expansibility coefficients have been assessed. In contrast to the denaturation of most globular proteins, the lysozyme denaturation under conditions of the experiment was accompanied by positive volume changes. Possible reasons for this unusual behavior have been discussed.     

Isolation of Extreme Halophiles from Seawater

Extreme halophilic bacteria were isolated from the ocean off the coast of Spain. All were gram-negative cocci. One isolate was compared to Halococcus sp. NCMB 757 and was found to have similar characteristics.     

缓步动物热溶性蛋白的独有特性及其在极端环境适应中的功能

缓步动物（tardigrades，俗称水熊虫）等一些低等动物可在干燥、低温、低压等极端条件下长期生存。这种超常的生存能力依赖于细胞在大幅度脱水后，进入一种叫做隐生（cryptobiosis）的特殊状态，使细胞脱水、身体萎缩并停止新陈代谢，从而可以允许动物在极端条件下生存多年。当环境好转时，处于隐生状态的细胞或者身体又可以再次吸收水份进行复苏。缓步动物中有着多种独特的内在无序蛋白质（intrinsic disorder protein），统称为热溶性蛋白质。这些热溶性蛋白质在细胞脱水过程中构象发生重要变化，可对液态水进行固定，从而起到了重要的细胞保护作用。对此类蛋白质的性质研究尚处于初期阶段，缺乏深入的机理性研究。本文简要总结了缓步动物中特有热溶性蛋白质的序列特征、理化性质，及其潜在的生物功能与机制。同时讨论了这些热溶性蛋白质在高等动物细胞对低温、低氧等极端环境适应中的可能应用。人类细胞在极端环境中的隐生和可逆复苏，将在医学领域和未来宇宙探索与星际移民中有极其重要的用途。     

嗜热菌——工业用酶的新来源

综述了嗜热菌和极端嗜热菌产生的热稳定性的淀粉酶、纤维素酶、环糊精酶、木聚糖酶、几丁质酶、葡萄糖异构酶、蛋白酶等的研究进展及其在食品、化工、环保等方面的应用前景。     

Hydrostatic Pressure and Proteins: Basic Concepts and New Data

In this paper we review some basic facts about the reversible and irreversible effects of high pressure on proteins. The effects include changes in intra- or intermolecular interactions (noncovalent bonds), in conformation and in solvation. Particular attention is directed to the interpretation of data where pressure-temperature dependency is an important phenomenon. Using model reactions, we have formulated a putative interpretation of physiological problems; we use these to explain how biological systems maintain activity when the temperature decreases and pressure increases, as in the case of barophilic micro-organisms in the deep sea world.     

STOP Proteins are Responsible for the High Degree of Microtubule Stabilization Observed in Neuronal Cells

Neuronal differentiation and function require extensive stabilization of the microtubule cytoskeleton. Neurons contain a large proportion of microtubules that resist the cold and depolymerizing drugs and exhibit slow subunit turnover. The origin of this stabilization is unclear. Here we have examined the role of STOP, a calmodulin-regulated protein previously isolated from cold-stable brain microtubules. We find that neuronal cells express increasing levels of STOP and of STOP variants during differentiation. These STOP proteins are associated with a large proportion of microtubules in neuronal cells, and are concentrated on cold-stable, drug-resistant, and long-lived polymers. STOP inhibition abolishes microtubule cold and drug stability in established neurites and impairs neurite formation. Thus, STOP proteins are responsible for microtubule stabilization in neurons, and are apparently required for normal neurite formation.